The structure of biological macromolecules is primarily detected using X-ray crystallography where the diffraction patterns of coherent X-rays interacting with crystallized biological macromolecules is recorded. To obtain highly resolved representations of the molecule's structure these diffraction patterns have to be recorded at wide angles where diffraction intensities are very low. The diffraction intensity is proportional to the number of diffracted X-rays and the number of unit cells in the illuminated single crystal. Therefore, in order to get sufficient diffraction intensities large crystals have to be irradiated with large numbers of photons.
The number of diffracted photons can easily be increased by extending the irradiation time. Unfortunately, many crystals only tolerate dose up to 30 MGy (3×107 J/kg) before substantial structural damages occur that destroy the crystalline structure. The dose required to obtain sufficient diffracted intensities can be reduced by growing larger crystals with more unit cells. For large macromolecules such as proteins and protein complexes staying below the tolerable dose requires crystals of many hundreds of micrometers in size. Growing crystals of this size and high quality is a very difficult and time consuming trial-and-error process that is currently the major bottleneck of X-ray crystallography. Even more challenging are membrane-bound proteins that are critical for drug design but notoriously difficult to crystallize.
The crystallization bottleneck can be overcome by using serial femtosecond crystallography. Here, nanocrystals are not irradiated with conventional synchrotron radiation but a collection of nanocrystals are irradiated one at a time with ultra-short coherent X-ray pulses. This method bears several advantages as the nanocrystals can be more easily grown and substantially higher doses can be employed.
Nanocrystals of many macromolecules can be grown by driving a protein suspension into supersaturation. If the proteins in the supersaturated suspension are quickly precipitated many small nanocrystals are formed around many nucleation sites.
The ultra-short coherent X-ray pulses are commonly generated with X-ray free electron lasers (X-ray FELs) and have a pulse length of approximately 100 fs (10-13 s). If the X-ray FEL pulse is focused to micrometer dimensions it can deposit doses in a crystal that exceed those conventionally tolerated by several magnitudes. As expected, the high dose of the X-ray pulse completely vaporizes the nanocrystal but only after the pulse has passed through it. The short pulse “outruns” the radiation damage as the inertia of the atoms in the crystal is sufficiently large to keep their movements within tolerable bounds during the time that the beam passes through the crystal. Hence, the diffraction pattern that is recorded on the detector corresponds to the undamaged crystal structure.
A single pulse does of course only give a diffraction pattern of the crystal structure in one particular orientation. In order to reconstruct the full three-dimensional structure of the molecule diffraction patterns obtained under many orientations have to be combined. Unlike conventional powder diffraction crystallography the data from many crystals is not summed up without regard to their orientation. Instead each diffraction pattern is indexed i.e. the observed peak intensities are labeled according to their origin in the lattice of the crystal. Those peaks that carry the same index are then summed up. The summation therefore averages over crystal shapes, crystal sizes and crystal orientation. Due to their small size the crystals are coherently illuminated which, combined with the index summation, leads to brighter intensities than those obtained with conventional crystallography on large crystals. It is therefore expected that more information can be extracted. It may, for example, be possible to extract a three dimensional vector gradient of the intensities which would increase the measured information by a factor of four. This would allow the use of novel phasing methods to obtain the molecular structure of the macromolecule.
In total, diffraction patterns from more than 10,000 crystals have to be measured and summed up. In these experiments approximately 10% of the X-ray pulses hit a nanocrystal. Out of the recorded diffraction patterns roughly half can be indexed successfully. Therefore, a total of 200,000 X-ray pulses is required to obtain sufficient data for a complete reconstruction of the structure of the macromolecule. Current X-ray FELs achieve a repetition rate of 120 Hz i.e. for each macromolecule at least 28 minutes of beam time is required.